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WO2011086990A1 - Cuve a circulation jetable de type puce et trieur de cellules utilisant ladite cuve - Google Patents

Cuve a circulation jetable de type puce et trieur de cellules utilisant ladite cuve Download PDF

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Publication number
WO2011086990A1
WO2011086990A1 PCT/JP2011/050270 JP2011050270W WO2011086990A1 WO 2011086990 A1 WO2011086990 A1 WO 2011086990A1 JP 2011050270 W JP2011050270 W JP 2011050270W WO 2011086990 A1 WO2011086990 A1 WO 2011086990A1
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WIPO (PCT)
Prior art keywords
flow
cell
flow path
fine particles
sample
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PCT/JP2011/050270
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English (en)
Japanese (ja)
Inventor
武田　一男
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株式会社オンチップ・バイオテクノロジーズ
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Publication date
Priority to EP22214423.0A priority Critical patent/EP4191227A1/fr
Priority to CN201180006138.7A priority patent/CN102753955B/zh
Priority to EP11732860.9A priority patent/EP2525209B1/fr
Priority to EP20156140.4A priority patent/EP3671180B1/fr
Priority to US13/521,947 priority patent/US10101261B2/en
Priority to JP2011549974A priority patent/JP5857357B2/ja
Application filed by 株式会社オンチップ・バイオテクノロジーズ filed Critical 株式会社オンチップ・バイオテクノロジーズ
Publication of WO2011086990A1 publication Critical patent/WO2011086990A1/fr
Priority to US14/923,747 priority patent/US10222317B2/en
Priority to US16/124,951 priority patent/US10724938B2/en
Priority to US16/194,315 priority patent/US10648899B2/en
Priority to US16/865,350 priority patent/US20200256785A1/en
Priority to US16/904,737 priority patent/US20200319084A1/en
Priority to US18/800,554 priority patent/US20240402068A1/en

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
    • G01N15/10—Investigating individual particles
    • G01N15/14—Optical investigation techniques, e.g. flow cytometry
    • G01N15/1434—Optical arrangements
    • G01N15/1436—Optical arrangements the optical arrangement forming an integrated apparatus with the sample container, e.g. a flow cell
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
    • B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
    • B01L3/502—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
    • B01L3/5027—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip
    • B01L3/502761—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip specially adapted for handling suspended solids or molecules independently from the bulk fluid flow, e.g. for trapping or sorting beads, for physically stretching molecules
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
    • G01N15/10—Investigating individual particles
    • G01N15/14—Optical investigation techniques, e.g. flow cytometry
    • G01N15/1484—Optical investigation techniques, e.g. flow cytometry microstructural devices
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2200/00—Solutions for specific problems relating to chemical or physical laboratory apparatus
    • B01L2200/06—Fluid handling related problems
    • B01L2200/0647—Handling flowable solids, e.g. microscopic beads, cells, particles
    • B01L2200/0652—Sorting or classification of particles or molecules
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00—Additional constructional details
    • B01L2300/08—Geometry, shape and general structure
    • B01L2300/0809—Geometry, shape and general structure rectangular shaped
    • B01L2300/0816—Cards, e.g. flat sample carriers usually with flow in two horizontal directions
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2400/00—Moving or stopping fluids
    • B01L2400/04—Moving fluids with specific forces or mechanical means
    • B01L2400/0475—Moving fluids with specific forces or mechanical means specific mechanical means and fluid pressure
    • B01L2400/0487—Moving fluids with specific forces or mechanical means specific mechanical means and fluid pressure fluid pressure, pneumatics
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2400/00—Moving or stopping fluids
    • B01L2400/06—Valves, specific forms thereof
    • B01L2400/0633—Valves, specific forms thereof with moving parts
    • B01L2400/0666—Solenoid valves
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2400/00—Moving or stopping fluids
    • B01L2400/08—Regulating or influencing the flow resistance
    • B01L2400/082—Active control of flow resistance, e.g. flow controllers
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
    • B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
    • B01L3/502—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
    • B01L3/5027—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip
    • B01L3/502738—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip characterised by integrated valves
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
    • B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
    • B01L3/502—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
    • B01L3/5027—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip
    • B01L3/502769—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip characterised by multiphase flow arrangements
    • B01L3/502776—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip characterised by multiphase flow arrangements specially adapted for focusing or laminating flows
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
    • G01N15/10—Investigating individual particles
    • G01N15/14—Optical investigation techniques, e.g. flow cytometry
    • G01N15/149—Optical investigation techniques, e.g. flow cytometry specially adapted for sorting particles, e.g. by their size or optical properties
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
    • G01N15/10—Investigating individual particles
    • G01N2015/1006—Investigating individual particles for cytology

Definitions

  • the present invention relates to a device having an analysis function of a biological particle such as a flow cytometer or a separation function such as a cell sorter, a measurement method that realizes a new function using the device, and a disposable flow cell chip.
  • Flow cytometers are generally widely used to differentiate between various types of cells and biological particles.
  • Conventional flow cytometers are typically equipped with an optically transparent flow cell made of quartz and having flow paths formed through it to allow the flow of cells to be individually identified.
  • the flow of cells through this flow path is concentrated in the center of the flow path by a sheath liquid that concentrically surrounds the flow of cells.
  • the central portion of this flow path is irradiated with a laser beam, and when the cells pass through the irradiation region, light scattering depending on the size, shape, and refractive index of the cells occurs.
  • the wavelength of the laser light is determined by the combination with the type of fluorescent dye in order to detect the cells specifically stained with the fluorescent dye with fluorescence.
  • Patent Document 1 A flat flow cell is described in JP-A-2003-302330 (Patent Document 10) and US Pat. No. 7,105,355 (Patent Document 11).
  • Patent Document 14 A flow cytometer using a disposable tip is described in Japanese Patent No. 4358888 (Patent Document 14).
  • Patent Document 1 U.S. Pat. No. 3,710,933
  • Patent Document 2 U.S. Pat. No. 3,826,364
  • Patent Document 3 Japanese Patent Application Laid-Open No. 64-3541
  • Patent Document 4 describes a method in which a pressure pulse is applied to particles flowing in a flow cell to separate the particles into a flow channel that is not a steady flow channel in the flow cell.
  • Patent Document 5 International Publication No. WO98 / 10267
  • Patent Document 6 discloses a method of separating cells charged in a liquid with an electric field by gel electrodes installed on both sides of a flow path in the flow cell.
  • Patent Document 7 a system is disclosed in which a pressure pulse is applied by a bubble valve that forms a meniscus perpendicular to the flow of particles to shift the flow for separation.
  • WO 2006/076195 Patent Document 8 discloses a method of ejecting water droplets containing particles for the purpose of applying a physical shock pulse as in Patent Document 5 and collecting them in a container.
  • Patent Document 9 when a particle in a flow of a sample liquid squeezed by a sheath flow is measured and determined to be a target particle, a pulse flow using a piezo element is disclosed. Describes a method of introducing into a separate flow path and separating.
  • Patent Document 12 The cell separation method described in Patent Document 12 is a method in which force is applied to droplets containing target cells by an electrostatic field by forming and flowing droplets containing cells in oil.
  • Patent Document 13 a method is used in which a branch flow path for cell sorting is formed in a flow path in a flow cell, a pulsed flow is generated using a piezo element, and necessary cells are drawn into the sorting flow path. Yes.
  • the flow path pattern can be easily and inexpensively mass-produced by injection molding or the like.
  • a flat flow cell When a flat flow cell is used, a method in which an irradiation laser is incident vertically from the surface is convenient.
  • side scattered light there is a problem in detecting scattered light in the in-plane direction of the flat plate, that is, side scattered light.
  • the significance of side scattered light detection is that information on the intracellular structure can be obtained, which is an essential function in a normal flow cytometer.
  • the flow cell of a normal flow cytometer generally has a square cross section, and there is no problem in measuring the scattered light in the direction perpendicular to the laser irradiation direction and it is measured simultaneously with the forward scattered light. .
  • Patent Document 11 describes a method in which an optical fiber is installed on a side surface of a flow path of a flow cell and light generated in the flow path is guided to a photodetector.
  • the optical fiber since the optical fiber is connected to the flow cell, it is not suitable for exchange of the flow cell for each measurement, and therefore cannot be applied to a flow cell for disposable use.
  • the flow cytometer using the disposable chip type flow cell of Patent Document 14 which is a previous invention by the present inventor does not include a side scattered light detection function.
  • the flow cell cannot be manufactured at a low cost, it becomes difficult to make it disposable.
  • it is convenient that it is made of a transparent resin.
  • the resin has a slight light absorption band in the wavelength region shorter than the wavelength of 500 nm and generates fluorescence, it becomes a background noise of measurement. That is, in the case of a flow cell made of transparent resin that is convenient as a disposable, autofluorescence becomes an obstacle to measurement.
  • the first issue is the problem of measures against biohazards.
  • a method (jet-in-air method) in which droplets are ejected by a jet nozzle described in Patent Document 1 or Patent Document 2 and droplets containing cells to be separated are separated by an electric field in units of droplets is a biohazard.
  • jet-in-air method There is a problem in measures. In other words, if the sample is a cell contaminated with pathogenic viruses or bacteria, there is a risk that a very dangerous substance is diffused into the atmosphere as an aerosol.
  • a method for solving the first problem a method of separating the aerosol while confined in the flow cell without diffusing into the atmosphere can be considered. As such a method, the following several techniques are disclosed.
  • Patent Document 3 discloses a method of separating and measuring by flowing a sheath flow around a sample liquid flowing through a flow cell and shifting an electric field from the sample flow to the sheath flow by applying an electric field to the sample liquid.
  • Patent Document 4 discloses a method of separating particles into a flow path that is not a steady flow path in the flow cell by applying a pressure pulse generated by a piezo element to the particles flowing through the flow cell. Then, since the separated particles do not return to the original flow path, complicated control such as the necessity to form a steady flow of air is a problem.
  • Patent Literature 5 a sheath flow is flowed around a sample liquid flowing in a flow cell to restrict the flow of the sample liquid, and an electric field or a magnetic field is applied to the fine particles in the sample liquid to shift and separate the flow of the fine particles.
  • the technology is disclosed.
  • an electric field is applied as a field in this method, it corresponds to the contents of Patent Document 3 above.
  • the method using these electric fields is the same as the method in Patent Document 6, and even if the generation of bubbles due to electrolysis is prevented by any method, the charge of the cells is shielded by the ions contained in the surrounding electrolyte, and the particles This is not practical for separation in an electrolyte because the force acting on the electrolyte is reduced.
  • Patent Document 7 discloses a cell separation technique in a chip. This is a method in which the flow of a single particle is shifted by the pressure from the side surface and separated downstream. However, the reciprocation of the meniscus is necessary to apply pressure, and a reverse flow occurs on the way back and forth, so it is necessary to restore the meniscus position after waiting for the particles to move sufficiently away.
  • Patent Document 8 discloses a method in which a physical shock pulse is applied and a region including a target cell is ejected in units of water droplets and collected in a container, as in Patent Document 4. This is not a content that can be realized in a disposable flow cell chip, and there is contamination with other samples. The method of Patent Document 9 cannot be applied to disposable chips.
  • Patent Document 12 is a method in which a droplet containing cells is formed in an oil and allowed to flow to separate charged droplets containing a target cell by applying an electrostatic force.
  • the oil does not have ions that shield electrostatic force, but there is a disadvantage that sorting of droplets larger than cells in oil with high viscosity is slow.
  • the method of generating a pulse flow using a piezo element and separating the cells into the branch flow paths is not premised on the connection between the flow cell and the piezo. If a piezo element is incorporated in the flow cell, the flow cell becomes expensive and is not suitable for a disposable chip.
  • a multi-color analysis of a flow cytometer generally performs fluorescence correction.
  • this fluorescence correction since a plurality of signal intensities distributed among a plurality of detectors having different detection wavelengths are corrected to one signal intensity for each fluorescent label, the apparatus is adjusted using a measurement sample before measurement. There is a need. Therefore, the sample for measurement needs the sample dye
  • the present invention provides an apparatus for analyzing and identifying biological particles using the following disposable chip type flow cell, and a disposable flow cell.
  • a flow cell in which a flow path is formed in a flat substrate; A light irradiation means for irradiating the fine particles in the sample liquid flowing through the flow path with light; Detecting means for detecting scattered light or fluorescence generated from the fine particles when irradiated with the light, identifying the fine particles based on their signal intensity, and detecting target fine particles; A constant pressure pump for applying a pressure pulse to the fine particles in the sample liquid flowing through the flow path of the flow cell, and an electromagnetic valve connected thereto, A control unit for controlling the operation of the electromagnetic valve based on a signal from the detection means,
  • the flow cell is A flow path for introducing the sample liquid and a pair of sheath liquid introducing flow paths disposed on both sides thereof; A merging channel formed by merging the pair of sheath fluid introduction channels with the sample solution introduction channel, wherein the sheath solution flows on both sides of the sample solution in the merging channel; A confluence channel; A light irradiation region on the confluence channel; A pair
  • the microparticles While passing through the region of the merged flow path to which the pair of opposed branch flow paths are connected, the first and second electromagnetic valves are given a signal to be opened for only a short time, thereby facing the opposite flow paths. Applying push (PUSH) and pull (PULL) pressure to the target fine particles passing through the region from a pair of branch channels, thereby changing the flow of the target fine particles.
  • a fine particle separation apparatus for separating fine particles. [2] The fine particle separator according to [1] above, When the control unit determines that the fine particles are target fine particles to be separated, the fine particles pass through the region of the merged flow channel to which the pair of opposed branch flow channels are connected.
  • the fine particle separator according to [1] above, The flow cell further includes a pair of additional branch channels that branch from the merge channel downstream of the region of the merge channel to which the pair of opposed branch channels connect, When it is determined that the fine particles are target fine particles to be separated, the control unit connects the first branch flow channel to which the first pair of branch flow channels face each other.
  • a fine particle separation apparatus for separating fine particles contained in a sample solution in a state where the sample solution is flowed through a flow path, The flow cell has a structure in which a flow path is formed in a transparent substrate, and a reservoir connected fluidly to the flow path (fluidly connected) is formed on the upstream and downstream sides of the flow path, respectively.
  • the flow cell apparatus is characterized in that the pair of branch flow paths includes ports that can be hermetically connected to an external pump.
  • a multi-sample compatible flow site for irradiating a sample liquid with a sample liquid containing biological fine particles flowing in a flow channel in the flow cell and detecting light generated from the sample particles contained in the sample liquid A meter, A stage on which the flow cell is placed, a light irradiation means, A detection means for detecting the biological microparticles, and a control unit for controlling the operation of each unit,
  • the flow cell has a flat plate shape, and a plurality of sample liquid reservoirs and a plurality of sheath liquid reservoirs, a plurality of waste liquid reservoirs and a plurality of recovered sample liquid reservoirs, and a plurality of flow paths fluidly connected to each of them on a transparent flat substrate Is formed, Each sample liquid reservoir is formed in each sheath liquid reservoir so as to be partitioned so that the respective liquids are not mixed, Each sample solution channel is connected to each sample solution reservoir, A pair of sheath fluid channels are connected to each of the sheath fluid reservoirs, The pair of sheath liquid
  • Multi-sample flow cytometer [6] The multi-sample compatible flow cytometer according to [5] above, The side scattered light generated from the inside of each flow path is totally reflected at both ends and detected by inclining both ends in the side surface direction of the flow path of the transparent flat substrate of the flow cell. Multi-sample flow cytometer.
  • an irradiation light source comprising a fluorescence measuring device configured to measure fluorescence in a plurality of wavelength regions, In the analysis of cells containing multiple fluorescent molecules, it has the function of deriving and displaying the distribution of individual fluorescence intensity ratios of two types of fluorescence intensities generated by light irradiation, A flow cytometer comprising a function of evaluating the abundance ratio of a plurality of fluorescent molecules contained in a cell based on the value.
  • a flow cell in which a flow path is formed in a flat substrate;
  • An irradiation light source for irradiating light in a state where fine particles in the sample liquid are flowed to the flow cell along with the sheath liquid in the flow path;
  • Detecting means for detecting scattered light or fluorescence generated from the fine particles when irradiated with the light, identifying the fine particles based on their signal intensity, and detecting target fine particles;
  • a liquid particle measuring apparatus comprising: a control / analysis unit that controls each unit and analyzes the particles based on information from the detection unit; Utilizing a signal representing bubbles generated when all of the sample liquid has flowed, the number of particles contained in the entire sample liquid is quantified by detecting the end point of the sample liquid measurement, Liquid particle measuring device.
  • an irradiation light source comprising: a light measurement device configured to measure light generated from individual particles by light irradiation in a plurality of wavelength regions; A biological particle analyzer having a function of obtaining an index for each particle by calculation between a plurality of signal intensities corresponding to the measurement item of the particle and analyzing particle characteristics based on the index value.
  • Bio microparticle analysis apparatus comprising: an arithmetic means for determining whether a cell is a dead cell or a living cell based on a ratio of two kinds of fluorescence signal intensities generated by light irradiation and having different wavelengths as an index. .
  • an irradiation light source comprising: a light measurement device configured to measure light generated from individual particles by light irradiation in a plurality of wavelength regions;
  • a biological particle analysis apparatus characterized in that, for each particle, an index defined by an arithmetic expression between a plurality of signal intensities can be derived, and the index can be selected as an axis of a data display graph.
  • conditions satisfying no carryover or cross-contamination are satisfied, 1) a cell sorter, 2) a flow cytometer capable of detecting side scattered light, 3) accurate cell density measurement, and 4) fluorescence correction. Unnecessary multiple staining analysis is realized.
  • (A) It is the figure which showed the subject in the case of performing cell sorting by a pulse flow in a flow path.
  • (B) It is the figure which showed the principle which solves said subject by this invention.
  • a) It is the figure which showed an example of the cell sorter by this invention, and has shown the state of electromagnetic valve OFF.
  • b) It is the figure which showed an example of the cell sorter by this invention, and has shown the state of electromagnetic valve ON.
  • a) It is the figure which showed the 2nd form of the cell sorter by this invention, and has shown the state of electromagnetic valve OFF.
  • b) It is the figure which showed the 2nd form of the cell sorter by this invention, and has shown the state of electromagnetic valve ON.
  • the present invention provides a particulate separator.
  • This particulate separator typically has a i) a flow cell having a flow path formed in a flat substrate; ii) a light irradiation means for irradiating the fine particles in the sample liquid flowing through the flow path with light; iii) detecting means for detecting scattered light or fluorescence generated from the fine particles when irradiated with light, identifying the fine particles based on their signal intensity, and detecting the target fine particles; iv) a constant pressure pump for applying a pressure pulse to the fine particles in the sample liquid flowing through the flow path of the flow cell, and an electromagnetic valve connected thereto, v) A control unit that controls the operation of the electromagnetic valve based on a signal from the detection means.
  • the flow cell is a) Sample liquid introduction flow path and a pair of sheath liquid introduction flow paths arranged on both sides thereof; b) a merging channel formed by merging the pair of sheath liquid introducing channels with the sample solution introducing channel [a sheath solution flows on both sides of the sample solution in the merging channel]; c) a light irradiation region on the confluence channel; d) A pair of opposed branch channels connected to the merge channel (substantially at right angles) downstream from the light irradiation region.
  • one of the pair of opposed branch channels is connected to a normally closed first electromagnetic valve and a negative pressure constant pressure pump, and the other of the pair of opposed branch channels is Is connected to a normally closed second solenoid valve and a positive pressure constant pressure pump.
  • the detection means detects an optical signal generated when the fine particles pass through the light irradiation region.
  • the control unit determines whether or not the fine particles are target fine particles to be separated based on the optical signal from the detection unit, and is determined to be the target fine particles to be separated.
  • the first and second electromagnetic valves are given a signal to be opened only for a short time while the fine particles pass through the region of the merged flow path where the pair of opposed branch flow paths are connected.
  • push (PUSH) and pull (PULL) pressures are applied to the target fine particles passing through the region from the pair of opposed branch channels. In this manner, the target fine particles can be separated from the other fine particles by changing the flow of the target fine particles.
  • the flow channel joins the pair of opposed branch flow channels. While passing through this region, a signal for opening the electromagnetic valve for only a short time is given, thereby pushing and pulling the target fine particles present in the region of the confluence channel between the opposed branch channels. Thus, the flow of the target fine particles may be changed, and the target fine particles may be taken into the pull-side branch channel.
  • the flow cell further includes a pair of additional branch channels that branch from the merge channel downstream of the region of the merge channel to which the pair of opposed branch channels connect. Also good.
  • the control unit determines that the fine particles are target fine particles to be separated, the fine particles are connected to the first branch flow channel where the first pair of branch flow channels face each other.
  • the electromagnetic valve While passing through the region, the electromagnetic valve is given a signal to open for only a short time, thereby pushing against the target particulates present in the region of the merging channel between the first opposing branching channels. And by applying a pull pressure and changing the flow of the desired particulates, the particulates can be separated by separating the desired particulates into an additional downstream branch.
  • the present invention provides a flat plate flow cell device for separating fine particles contained in a sample solution in a state where the sample solution is passed through a flow path.
  • a flow path is formed in a transparent substrate, and a reservoir fluidly connected to the flow path is formed on each of the upstream and downstream substrates of the flow path. Has a structure.
  • this flow cell apparatus is formed on a transparent substrate. i) a flow path for introducing a sample liquid and a pair of flow paths for introducing a sheath liquid disposed on both sides thereof; ii) A merging channel formed by joining the channel for introducing the sample liquid and the pair of sheath liquid introducing channels disposed on both sides of the channel, and the sheath liquid on both sides of the sample liquid.
  • the pair of branch flow paths is provided with a port that can be hermetically connected to an external pump.
  • the flow cell structure may include a pair of additional branch channels that branch from the junction channel downstream of the region of the junction channel to which the branch channel connects.
  • the present invention is directed to irradiating a sample liquid with a sample liquid containing biological microparticles in a flow channel in a flow cell, and from the sample particles contained in the sample liquid.
  • a multi-analyte flow cytometer for detecting generated light. This flow cytometer is typically i) A stage on which the flow cell is placed ii) light irradiation means; iii) detection means for detecting biological microparticles; and iv) a control unit that controls operations of the above-described units.
  • the flow cell may be flat. Further, the flow cell has a plurality of sample liquid reservoirs and a plurality of sheath liquid reservoirs, a plurality of waste liquid reservoirs and a plurality of recovered sample liquid reservoirs formed on a transparent flat substrate, and a plurality of flow paths for fluid connection therewith. May be. Further, each sample liquid reservoir may be formed in each sheath liquid reservoir so as to be partitioned so that the respective liquids are not mixed. Further, each sample solution channel may be connected to each sample solution reservoir. Further, a pair of sheath fluid channels may be connected to each of the sheath fluid reservoirs. Furthermore, the pair of sheath liquid flow paths may be connected to the side surfaces of the sample liquid flow paths.
  • a flow path in which the sheath flow is merged from the left and right of the sample liquid flow is formed, and the merged flow paths are arranged at equal intervals in parallel. It is formed, and the most downstream may have a structure connected to the waste liquid reservoir and the recovered sample reservoir formed on the flow cell.
  • the irradiation light or the flow cell is sequentially moved in a step-and-repeat manner with a light beam of a size that is irradiated to only one of the plurality of channels.
  • a plurality of samples can be measured.
  • the side scattered light generated from each of the flow paths is generated at both ends by inclining the both ends in the lateral direction of the flow paths of the transparent flat substrate of the flow cell. And may be totally reflected and guided to a detection system.
  • Flow Cytometers in yet another embodiment of the present invention, in the analysis of a cell containing a plurality of fluorescent molecules, the ratio of two types of fluorescence intensities generated by laser light irradiation with different wavelengths is distributed and displayed for each cell.
  • a flow cytometer characterized by having a function and a function of evaluating the abundance ratio of a plurality of fluorescent molecules contained in a cell according to the value.
  • the ratio of two types of fluorescence signal intensities generated by laser light irradiation with different wavelengths is used.
  • a biological particle analyzing apparatus that determines whether a cell is a dead cell or a living cell based on a magnitude relationship with respect to a preset reference value.
  • These flow cytometers or biological particle analyzers typically include at least a laser light irradiation source and a fluorescence measurement device configured to measure fluorescence in a plurality of wavelength regions.
  • FIG. 1A illustrates a flow caused by pressure when a pressure is applied from a cell sorting flow channel 100 connected to a main flow channel through which cells flow.
  • PULL state the flow is drawn (PULL state).
  • PUSH state the flow is pushed out (PUSH state). It is impossible to move only the target cell by applying the pressure of PULL or PUSH at the moment when the target cell flows through the main channel and crosses the sorting channel. That is, when only specific cells are separated into the separation flow path by pressure, the force reaches a wide range of liquids, resulting in poor separation spatial resolution.
  • the PUSH and PULL sorting channels are arranged to face each other on the side surface of the main channel, and usually the main channel and the sorting channel
  • the pressure of PUSH and the pressure of PULL are generated only during the time when the target cells to be separated pass through the opposed regions.
  • the flow rate of PUSH and the flow rate of PULL are made substantially the same, and the region where the pressure from the sorting channel affects the fluid of the main channel is almost limited to the sorting channel width. This method is shown in FIG.
  • the sample solution containing cells flows through the flow channel 1 formed in the flow cell, the sheath solution containing no cells flows through the flow channel 2, and the sample solution flows in a narrowed state by joining.
  • Two branch channels 4-1 and 4-2 are connected opposite to the side surface of the channel through which the sample flows.
  • the channel 4-1 is a PULL channel
  • the channel 4-2 is a PUSH channel.
  • a reservoir 5 for storing target cells, an electromagnetic valve 7-1, and a cylinder pump 8-1 are connected to the flow path 4-1.
  • the cylinder pump 8-1 maintains a constant pressure sufficiently lower than the flow path 1, while the electromagnetic valve 7-2 and the cylinder pump 8-2 are connected to the PUSH flow path.
  • the cylinder pump 8-2 maintains a constant pressure sufficiently higher than the flow path 1.
  • scattered light and fluorescence are generated from the cells.
  • the light is detected by a photodetector, the signal intensity is quantified, and compared with a preset signal condition of necessary cells.
  • the signal processing circuit determines whether or not the cell is a necessary cell. If the cell is a necessary cell, a trigger signal is generated at the time when the cell passes through the branch flow path.
  • And 7-2 are set to the OPEN state only for a short time.
  • the same flow rate as that taken into the separation channel 4-1 from the main channel is supplied from the channel 4-2 to the main channel, so that the channel 4-1 and the channel 4-2 are connected.
  • the sorting flow is limited to prevent the spatial resolution of cell separation from becoming worse than the width of the channel 4-1.
  • a sorting pressure and a sorting generation time width are controlled independently by connecting a constant pressure pump and an electromagnetic valve.
  • the constant pressure pump is suitable as a biohazard because it does not generate aerosol.
  • FIG. 3 shows a method of separation when the flow rate by PUSH & PULL is slower than the flow rate of the flow path 27 and cannot be taken into the sorting reservoir 5.
  • FIG. 3a) shows the case where the electromagnetic valve is OFF. On the downstream side, channels 11, 23 and 24 are formed symmetrically with channels 1 and 2. Due to the hydrodynamic effect in the laminar flow, the sheath liquid is separated into the flow paths 23 and 24, and the sample liquid 1 is collected in the flow path 11.
  • FIG. 3b) shows the case where the electromagnetic valve is ON. In this case, the flow of the target cell is generated by the pressure from the sorting channels 4-1 and 4-2, and the position slightly shifts from the channel 4-1 and flows through the channel 27. On the downstream side, the target cell is separated into the flow path 23 by this shift.
  • installing a filter to prevent cells and bacteria from flowing into the electromagnetic valve can help prevent bacteria and cells from entering the flow path from the outside of the flow cell and prevent the sample from escaping to the outside of the flow cell. it can.
  • FIG. 4 shows a state where a cylinder pump and an electromagnetic valve are connected to a disposable chip type flow cell.
  • the disposable chip is symmetrical to the sorting channels 4-1 and 4-2 and the sorting reservoirs 5-1 and 5-2 shown in FIG. It is assumed that the structure is added.
  • the disposable chip type flow cell is made of a transparent resin.
  • As the kind of resin polymetal acrylate (PMMA), cycloolefin copolymer (COC), methylpentene polymer, and the like can be used.
  • PMMA polymetal acrylate
  • COC cycloolefin copolymer
  • methylpentene polymer and the like can be used.
  • methylpentene polymer is suitable as a material for the flow cell.
  • a solenoid valve and a cylinder pump are connected as a symmetrical structure in which a sorting reservoir is also installed in the flow path 4-2.
  • the symmetrical structure is for the same pressure to be applied to the flow path 27 at the same time when the electromagnetic valve is opened.
  • Fig. 5 further shows the connection between the optical system and the control system circuit.
  • the cross-sectional view of the chip is the BB cross section of FIG.
  • the sample liquid flows along with the sheath liquid to the flow path 27, and the main flow slightly upstream from the area of the opposing sorting flow paths 4-1 and 4-2.
  • a laser beam 52 is applied to the center of the road. Scattered light and fluorescence are generated in a pulse manner at the moment when the cell passes through the irradiation region.
  • the scattered light is detected by the detector 61 by selecting the same wavelength as the irradiation laser light by the dichroic mirror 54 and the band pass filter 57.
  • a light shielding plate 60 is installed in front of the detector 61 to remove laser transmitted light.
  • the fluorescence is detected by different detectors 62 and 63 by dividing a plurality of wavelength regions longer than the irradiation laser light by the dichroic mirrors 55 and 56 and the bandpass filters 58 and 59, respectively.
  • Each detection pulse signal is digitized by a circuit 64 that amplifies and AD-converts, and a microcomputer 69 determines whether or not a plurality of detection signals satisfy a predetermined separation condition.
  • the trigger signal is output after a certain delay time. This delay time is adjusted to the time until the cells flow from the laser irradiation region to the sorting channels 4-1 and 4-2.
  • the electromagnetic valve driver 66 that has received the trigger signal outputs a signal for OPEN to the electromagnetic valves 7-1 and 7-2 for a predetermined time.
  • the time width when the valve is in the OPEN state is set to W / V (seconds) where the width of the flow path 4-1 and the flow path 4-2 is equal to W, and the flow rate of the cells through the flow path 27 is V. Is desirable.
  • the target cell flows into the sorting reservoir 5 at the time of the electromagnetic valve OPEN. Since the flow between the main flow path and the sorting flow path does not occur when the valves 7-1 and 7-2 are in the CLOSE state, the cells once taken into the sorting reservoir 5 are stably stored on the spot.
  • a constant air pressure is applied to the inside of the reservoir 20 on the upstream side of the chip by a constant pressure pump (not shown).
  • a sample solution reservoir 10 inside the reservoir 20, and a sample solution is put inside it, and a sheath solution is put outside it.
  • PBS phosphate buffer buffer
  • the width of the flow path 27 is 80 ⁇ m and the depth is 50 ⁇ m.
  • the width of the sample solution after the merge is about 1/10 of the channel width.
  • the channel 27 includes sorting channels 4-1 and 4-2 that are opposed from the side, and the channels are connected to the reservoir 5.
  • a laser beam 52 having a wavelength of 488 nm is applied to the central portion of the flow path 27 on the upstream side by several hundred ⁇ m from the facing region.
  • the beam size is an ellipse with a length of 50 ⁇ m and a width of 20 ⁇ m.
  • Scattered light and fluorescence are generated in a pulse manner at the moment when the cell passes through this irradiation region.
  • the scattered light is detected by the detector 61 by selecting the same wavelength as the irradiation laser light by the dichroic mirror 54 and the band pass filter 57.
  • a light shielding plate 60 is installed in front of the detector 61 to remove laser transmitted light.
  • the fluorescence is detected by different detectors 62 and 63 by dividing a plurality of wavelength regions longer than the irradiation laser light by the dichroic mirrors 55 and 56 and the bandpass filters 58 and 59, respectively.
  • Each detection pulse signal is digitized by a circuit 64 that amplifies and AD-converts, and a microcomputer 69 determines whether or not a plurality of detection signals satisfy a predetermined separation condition.
  • the trigger signal is output after a certain delay time. This delay time is the same as the time required for the cells to flow from the laser irradiation region to the sorting channels 4-1 and 4-2.
  • the electromagnetic valve driver 66 that has received the trigger signal outputs a signal for OPEN to the electromagnetic valves 7-1 and 7-2 for a predetermined time.
  • the time width when the valve is in the OPEN state is set to W / V (seconds) where the width of the flow path 4-1 and the flow path 4-2 is equal to W, and the flow rate of the cells through the flow path 27 is V.
  • the target cell flows into the sorting reservoir 5-1 at the time of the electromagnetic valve OPEN. Since the flow between the main flow path and the sorting flow path does not occur when the valves 7-1 and 7-2 are in the CLOSE state, the cells once taken into the sorting reservoir 5-1 are stably stored on the spot.
  • the flow cell shown in FIG. 4 has the separation channels 11, 23, and 24 formed on the downstream side, so that the target cell is subjected to the sorting reservoir 5 ⁇ by the pressure of the sorting channels 4-1 and 4-2. When there is no movement amount enough to be taken into 1, the flow is separated into the flow path 23 on the downstream side. Therefore, the flow cell shown in FIG. 4 has a wide margin in two stages with respect to the flow rate of separation into the flow channel 23 when the flow rate of the flow channel 27 is high, and separation into the sorting reservoir 5-1 when the flow rate is low. It has a structure that realizes separation.
  • FIG. 6 An example of a flow cytometer using a disposable chip type multi-channel flow cell is shown in FIG. And will be described with reference to FIG.
  • FIG. 6 In order to measure a large number of specimens under the condition of no contamination and zero carryover, as shown in FIG. 6, eight sample flow paths are formed in one chip, and a sample liquid scissor bar 31 connected to each of them is provided. Connected.
  • the eight chambers 37 partitioned so as not to mix in the reservoir 30 are sheath fluid reservoirs.
  • One sample reservoir is installed in each room. There are two ports each connecting the sheath fluid reservoir to the sheath fluid flow path.
  • the flow of the sheath liquid and the sample liquid is controlled by controlling the flow of the sample liquid and the sheath liquid connected to each room by sequentially applying air pressure to each room, by pressurizing only the room containing the sample liquid to be measured, Eliminates consumption due to wasteful flow of sample liquid and sheath liquid.
  • the channel cross section is the same size as the channel cross section of FIG.
  • the pressure during measurement is set to a value between 10 kPa and 20 kPa, and the pressure other than during measurement is the same atmospheric pressure as the downstream part. Inside each room, a common air pressure is applied to the sheath liquid and the sample liquid and pushed downstream.
  • FIG. 7 shows a cross-sectional view of this chip and a measurement optical system.
  • the detection optical system using the lens 51 in the measurement optical system is almost the same as that in FIG. 5, but an optical system for observing the image of the flow path is added. They are a 1% reflecting mirror, an imaging lens 81, and a camera 82.
  • the irradiation optical system including the laser and the detection optical system are fixed, and a system that measures eight channels by moving an automatic stage on which a chip is mounted is adopted.
  • the movement distance for the eight channels is set to 5 mm due to the restriction between the channels in manufacturing.
  • the stage is moved by aligning the irradiation position of the laser with the No. 1 flow path at the end by image recognition.
  • the upstream reservoir to which the No. 1 flow path is connected is pressurized at a constant pressure, and the sample solution is allowed to flow only in the No. 1 flow path to start measurement.
  • the pressure applied to the upstream reservoir connected to No. 1 is set to zero and the atmospheric pressure is set.
  • the stage is moved to align the laser irradiation position with the No. 2 channel, pressurize the reservoir connected to the No. 2 channel, and start measurement.
  • the side scattered light being measured reflects the end surface of the flat substrate downward with a slope of about 45 degrees, enters the light guide block 45 formed of a transparent resin, and is connected to the light guide block. Only the light having the same wavelength as the irradiated laser beam is detected by the photo detector 44 by the band pass filter 43 via.
  • the side scatter signals collected and detected by the light guide blocks at both ends are combined and measured.
  • FIG. 7 shows a method in which the measured scattered light at both ends is detected by two detectors and converted into a signal, and then the sum is measured.
  • the optical fibers connected to the light guide blocks at both ends may be measured by joining them to one detector.
  • the width of the light guide block 45 is set to 10 mm because it is necessary to make it larger than the moving distance of 8 channels in the chip of 8 channels.
  • FIG. 6 shows a cross-sectional view of this chip and a measurement optical system.
  • the detection optical system using the lens 51 in the measurement optical system is almost the same as that in FIG. 5, but an optical system for observing the image of the flow path is added. They are a 1% reflecting mirror, an imaging lens 81, and a camera 82.
  • the position adjustment between the laser position and the flow path can be automatically performed by grasping the relationship between the flow path and the laser irradiation position by image recognition and feeding back to the stage movement.
  • the laser beam and the detection optical system are fixed, and the center area of each flow path is sequentially irradiated onto the chip by step-and-repeat scanning on the automatic stage.
  • Side scattered light reflects down the end face of the flat substrate with a slope of about 45 degrees, enters the light guide block 45 made of transparent resin installed below, and passes through the optical fiber connected to the light guide block. Then, only the light having the same wavelength as the irradiation laser beam is transmitted by the band pass filter 43 and detected by the photodetector 44.
  • FIG. 8 a shows the positional relationship between the chip and the light guide blocks at both ends when laser light is irradiated to the center of the leftmost channel among the plurality of channels.
  • FIG. 8b shows the positional relationship between the chip and the light guide blocks at both ends when laser light is irradiated to the center of the rightmost channel among the plurality of channels.
  • the width of the light guide block in the scanning direction is larger than the distance of the scanning range so that the side scattered light reflected at both ends is incident on the light guide block.
  • the optical fibers connected to the light guide blocks at both ends may be directly joined to one detector.
  • FIG. 9a) shows a measurement of a cell derived from breast cancer called MCF-7, which contains bubbles because the whole sample solution was measured.
  • MCF-7 a cell derived from breast cancer
  • the air pushed from the sample reservoir becomes bubbles without passing through the flow path, passes through the measurement region, and increases the count.
  • the sample liquid disappears from the distribution larger than the cells (the forward scattering signal FS is large)
  • the A distribution in FIG. 9a) is generated. It can be seen that if the data after the detection time is deleted from the measurement data after the measurement, the A distribution can be removed while leaving the cell distribution and foreign matters other than the cells. This is because the A distribution is a distribution derived from bubbles generated after the time when the sample liquid disappears in the latter half of the measurement. Furthermore, the distribution removed together with the A distribution can be determined to be a bubble-derived distribution.
  • the flow path is connected to the lower side of the sample solution reservoir. If 100 microliters is placed in the sample solution reservoir and all the sample solution is measured, and the particle count is 10,000, the concentration of particles should be able to be derived as 100,000 particles / milliliter. However, this is not possible with conventional methods. That is, immediately after the sample liquid is exhausted, bubbles having a distribution different from that of particles are frequently generated, and this bubble distribution becomes an obstacle to counting particles. In a normal flow cytometer, a method of sucking up a sample solution from above into a sample solution container is usually used, and therefore the total amount of the sample solution cannot be measured or cannot be performed.
  • FIG. 9a) shows data including bubble distribution obtained by measuring the entire sample solution with the chip described in Patent Document 14 which is an invention of the present inventor.
  • the horizontal axis is forward scattering
  • the vertical axis is fluorescence signal intensity.
  • FIG. 9b) shows data obtained by removing the portion after the detection time from all the data. It can be seen that the distribution with large forward scattered light intensity is removed. If one pays attention to one of the bubble-derived distributions and removes data from the end of detection until the distribution disappears, the bubbles can be removed. As a result, the entire sample solution can be measured, and an accurate particle concentration can be obtained.
  • FIG. 10A shows the wavelength regions of two types of fluorescence detection signals FL1 and FL2 and the spectra of two types of fluorescent molecules A and B. Yes.
  • a method for obtaining the abundance ratio of A and B molecules in the cell when the cells are fluorescently stained with two types of fluorescent molecules A and B using the fluorescence signals of FL1 and FL2 will be described.
  • a relationship defined by the shape of the fluorescence spectrum exists between the fluorescence detection signals FL1 and FL2. That is the relationship that the ratio is constant.
  • the detection signal intensity ratio between FL1 and FL2 is A1 / A2.
  • Equation 8 [(B1 / B2)-(FL1 / FL2)] / [(FL1 / FL2)-(A1 / A2)] (B2 / A2) (8)
  • the first term on the right side of Equation 8 is that B1 / B2 is a constant obtained from sample data stained with 100% B, A1 / A2 is a constant obtained from sample data stained with 100% A, and FL1 / FL2 is the sample data. Measurement data.
  • the second term is a constant obtained by the ratio of FL2 of samples stained separately with A and B with the same number of molecules. Therefore, the value of x / y can be obtained for each detected cell. As shown in FIG.
  • FIG. 12 shows a conventional analysis method in the case of performing life / death determination analysis using two types of nuclear stains, ie, a nuclear stain SYTO9 that penetrates the biological membrane and a nuclear stain PI that does not penetrate the biological membrane.
  • an index is defined for life / death determination, and life / death determination is automatically performed based on a threshold set by the index. This method will be described with reference to FIGS.
  • the wavelength region of FL1 is about 500 nm to 550 nm
  • the wavelength region of FL2 is 570 nm to 610 nm. As shown in the distribution of FIG.
  • the conventional LIVE & DEAD analysis distinguishes a LIVE distribution (a distribution stained only with SYTO9) and a DEAD distribution (a distribution stained with both SYTO9 and PI).
  • This distinction method relies on human image recognition. Therefore, in the present invention, a quantitative index is created for automatic determination of LIVE and DEAD, and a determination threshold is set in the index to distinguish between LIVE and DEAD.
  • staining is performed with the following protocol with two types of SYTO9, which is a nuclear stain that permeates the cell membrane, and PI, which is a nuclear stain that does not permeate.
  • Fig. 12 shows the result of measurement by staining cells called MCF-7 with this treatment.
  • the LIVE distribution is a distribution of cells stained only with SYTO9 and shows one line distribution.
  • the DEAD distribution is double-stained because it is stained with PI due to the destruction of the cell membrane.
  • the ratio between the SYTO9 staining amount and the PI staining amount is almost constant. For this reason, the position of the DEAD distribution is a linear distribution shifted downward from the position of the LIVE distribution.
  • FIG. 13 is a graph showing the effect of an antibacterial agent against Pseudomonas aeruginosa by the above method. This is a histogram distribution obtained by measuring FL1 / FL2 after staining with SYTO9 and PI for 20 minutes after mixing with an antibacterial agent.
  • the threshold value for the determination of viability in the value shown by the broken line in FIG. 13 is a value set at the boundary of the distribution obtained by separately measuring 100% viable bacteria and 100% dead bacteria in advance. Life and death can be determined. Therefore, it can be seen that FL1 / FL2 is effective as an index for automatic life / death determination.
  • FIG. 14 shows the same measurement result as a scatter diagram using the life-and-death determination index of the present invention, compared to the scatter diagram of FIG.
  • the vertical axis is FL1 / FL2 and the horizontal axis is FS, which is a forward scatter intensity signal indicating the cell size, thereby realizing cell life and death information and cell size information in a single two-dimensional scatter diagram.
  • the scatter diagram of FIG. 14 shows the result that the dead cell is smaller in size than the living cell for the MCF-7 cell as the measurement sample.
  • FIG. 15 shows the measurement data of a sample of two types of cell surface markers, one stained with FITC fluorescently labeled antibody and the other stained with PE fluorescently labeled antibody, with PE / (FITC + PE) as the vertical axis, It is a display example that employs FS as an axis.
  • PE and FITC are fluorescence amounts after fluorescence correction, and are amounts proportional to the number of fluorescent molecules.
  • This index is defined as an amount indicating the expression ratio of two kinds of surface markers
  • FIG. 15 is a display example showing the cell size dependency of the expression ratio of two kinds of surface markers in one graph.
  • the excess or deficiency of the expression ratio of each cell can be analyzed for each cell.
  • (signal pulse area) / (signal pulse time width) or the like can be defined as an index for evaluating whether a detected cell is a single cell or a plurality of aggregated cells. Although floating cells such as leukocytes flow in a single cell state, cancer cells in the blood do not always circulate in the blood while remaining single. In such an evaluation, it is necessary to evaluate the number of cells regardless of whether they are aggregated or non-aggregated.
  • This index is an index useful for the analysis of the aggregation or non-aggregation. As described above, there are many merits to define an index that is convenient for analysis contents by calculation between a plurality of signals and quantitatively evaluate the characteristics of each cell using the index value.
  • the advantage is that multidimensional information can be combined into a single index, so that information from more than three types of signals can be expressed in a single two-dimensional scatter diagram, and a threshold value can be set in advance for that index. To achieve quantitative determination of individual cells. This method is effective for quantitative medical diagnosis.
  • a cell sorter there is no carryover or cross-contamination, 1) a cell sorter, 2) a flow cytometer capable of detecting side scattered light, 3) an accurate cell density measuring device, and 4) multiple staining analysis that does not require fluorescence correction. It is useful as a device and a cell separation / analysis method using these devices.
  • waste liquid reservoir 35 ... recovered sample liquid reservoir 36 ... sample liquid 37 ... sheath liquid reservoirs 39-1 and 39-2 ... reflective surface 40 for side scatter detection ... laser beam scanning region 41 ... flow path 50 ... Laser light source 51 ... Objective lens 52 ... Laser beam 53 ... Area 54, 55, 56 sandwiched between sorting channels 4-1 and 4-2 ... Dichroic mirror Over 57, 58, 59 ... band-pass filter 60 ... transmitted laser beam blocking spatial filter 61 ... conversion photodiode 62, 63 ... photomultiplier tube 64 ... AD unit 69 ... AD converter 70 ... keyboard 71 ... Display

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Abstract

L'invention concerne un séparateur de particules comprenant un trieur de cellules qui empêche un transport et une contamination croisée, et un cymomètre de flux qui peut détecter une lumière rétrodiffusée; et permettant de mesurer une densité cellulaire précise et d'effectuer une analyse multi-couleur qui ne requière par de compensation de fluorescence. Le séparateur de particules précité comprend une cuve à circulation dotée d'un canal formé dans un substrat plat; un moyen d'éclairage qui envoie de la lumière sur les particules microscopiques d'un liquide d'échantillon s'écoulant via le canal; un moyen de détection qui détecte la lumière ou la fluorescence dispersée provenant des particules lors de l'éclairage et identifie lesdites particules en fonction des puissances des signaux, ce qui permet de détecter les particules cibles; une pompe à pression constante qui applique des impulsions de pression sur les particules du liquide d'échantillon s'écoulant via le canal dans la cuve à circulation; une soupape électromagnétique connectée à la pompe à pression constante; et un moyen de commande qui commande la soupape électromagnétique en fonction d'un signal provenant du moyen de détection.
PCT/JP2011/050270 2010-01-15 2011-01-11 Cuve a circulation jetable de type puce et trieur de cellules utilisant ladite cuve WO2011086990A1 (fr)

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JP2011549974A JP5857357B2 (ja) 2010-01-15 2011-01-11 使い捨てチップ型フローセルとそれを用いたセルソーター
CN201180006138.7A CN102753955B (zh) 2010-01-15 2011-01-11 一次性芯片型流动室与利用其的细胞分选仪
EP11732860.9A EP2525209B1 (fr) 2010-01-15 2011-01-11 Cuve a circulation jetable de type puce et trieur de cellules utilisant ladite cuve
EP20156140.4A EP3671180B1 (fr) 2010-01-15 2011-01-11 Procédé d'utilisation d'un appareil configuré pour séparer des particules biologiques
US13/521,947 US10101261B2 (en) 2010-01-15 2011-01-11 Disposable chip-type flow cell and cell sorter using the same
EP22214423.0A EP4191227A1 (fr) 2010-01-15 2011-01-11 Cellule d'écoulement de type puce jetable et cytomètre de flux l'utilisant
US14/923,747 US10222317B2 (en) 2010-01-15 2015-10-27 Method for sorting cell particle in solution
US16/124,951 US10724938B2 (en) 2010-01-15 2018-09-07 Disposable chip-type flow cell and cell sorter using the same
US16/194,315 US10648899B2 (en) 2010-01-15 2018-11-17 Method for sorting cell particle in solution
US16/865,350 US20200256785A1 (en) 2010-01-15 2020-05-02 Disposable chip-type flow cell and flow cytometer using the same
US16/904,737 US20200319084A1 (en) 2010-01-15 2020-06-18 Disposable chip-type flow cell and cell sorter using the same
US18/800,554 US20240402068A1 (en) 2010-01-15 2024-08-12 Disposable chip-type flow cell and cell sorter using the same

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JP2010-007295 2010-01-15

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US14/923,747 Continuation US10222317B2 (en) 2010-01-15 2015-10-27 Method for sorting cell particle in solution
US16/124,951 Continuation US10724938B2 (en) 2010-01-15 2018-09-07 Disposable chip-type flow cell and cell sorter using the same

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Cited By (11)

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CN102998243A (zh) * 2012-11-26 2013-03-27 长春迪瑞医疗科技股份有限公司 一种抑制粒子翻滚的粒子成像装置及方法
WO2013146993A1 (fr) 2012-03-28 2013-10-03 株式会社オンチップ・バイオテクノロジーズ Procédé de détection du degré de malignité d'une unité de cellule tumorale circulante et trousse pour celui-ci
JP2015531062A (ja) * 2012-08-01 2015-10-29 アウル バイオメディカル インコーポレイテッドＯｗｌ Ｂｉｏｍｅｄｉｃａｌ， Ｉｎｃ． サイトメトリー機能を備えた粒子操作装置
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US20200319084A1 (en) 2020-10-08
EP2525209A1 (fr) 2012-11-21
JP2016057309A (ja) 2016-04-21
US10648899B2 (en) 2020-05-12
US10101261B2 (en) 2018-10-16
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EP4191227A1 (fr) 2023-06-07
CN104549584B (zh) 2016-11-30
US20160139026A1 (en) 2016-05-19
US20120288920A1 (en) 2012-11-15
US10222317B2 (en) 2019-03-05
US20240402068A1 (en) 2024-12-05
US20190154563A1 (en) 2019-05-23
US20190078995A1 (en) 2019-03-14
EP3671180A1 (fr) 2020-06-24
JP5857357B2 (ja) 2016-02-10
US20200256785A1 (en) 2020-08-13
JP6031178B2 (ja) 2016-11-24
CN102753955B (zh) 2015-02-04
EP3671180B1 (fr) 2023-03-08
CN102753955A (zh) 2012-10-24

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